Methods for Producing DNA Libraries and Uses Thereof

Abstract

Disclosed herein are methods for producing DNA libraries by incorporating dUTPs into DNA fragments and treating with uracil-DNA glycosylase and kits for preparing the DNA libraries. The DNA libraries are advantageous for next generation sequencing.

What is claimed is: 
     
         1 . A method of preparing a DNA library, comprising
 (a) ligating an adapter to a target DNA molecule to generate a ligation product, wherein the adapter comprises one or more deoxyuridines and a unique molecular index (UMI);   (b) treating the ligation product with a uracil-DNA glycosylase (UDG); and   (c) amplifying the target DNA molecule attached to the adapter in the ligation product of (b) by single primer extension with a target specific primer to generate a target amplification product, wherein the target specific primer comprises one or more deoxyuridines and a target specific sequence.   
     
     
         2 . The method of  claim 1 , wherein the target DNA molecule has been end-repaired. 
     
     
         3 . The method of  claim 1 or 2 , wherein the target DNA molecule has been adenylated. 
     
     
         4 . The method of any one of  claims 1-3 , wherein the target specific sequence comprises one or more deoxyuridines. 
     
     
         5 . The method of any one of  claims 1-4 , wherein the target specific primer comprises a tag sequence comprising at least 2 deoxyuridines and a target specific sequence comprising 0-4 deoxyuridines in a 5′ to 3′ direction. 
     
     
         6 . The method of any one of  claims 1-5 , further comprising treating the target amplification product with a UDG and then amplifying the target amplification product with a universal primer to generate a second target amplification product, wherein the universal primer does not comprise a deoxyuridine. 
     
     
         7 . The method of  claim 6 , wherein the universal primer further comprises a sample index. 
     
     
         8 . The method of any one of  claims 1-7 , wherein the ligating is performed at a pH of 8-9. 
     
     
         9 . The method of  claim 8 , wherein the ligating is performed at a PEG concentration of 4% PEG to less than 10% PEG. 
     
     
         10 . The method of any one of  claims 1-9 , wherein the amplifying is performed with an antibody based thermostable polymerase. 
     
     
         11 . The method of any one of  claims 6-10 , further comprising purifying the second target amplification product with beads. 
     
     
         12 . The method of  claim 11 , wherein the beads are solid phase reversible immobilization (SPRI) beads. 
     
     
         13 . The method of any one of  claims 1-12 , wherein the DNA library can be used for next generation sequencing, profiling of DNA variants, human identity or paternity testing, pain or ADME pharmacogenomics, or detection of a genetic disease. 
     
     
         14 . A method of preparing a DNA library, comprising amplifying a target DNA molecule with a single target specific primer to generate a target amplification product, wherein the target specific primer comprises a tag sequence, one or more deoxyuridines, and a target specific sequence. 
     
     
         15 . The method of  claim 14 , wherein the adapter is a single adapter that is annealed to the target DNA molecule and extended, wherein the adapter comprises a UMI and a target specific sequence that does not contain a deoxyuridine. 
     
     
         16 . The method of  claim 14 , wherein a pair of adapters are annealed to the target DNA molecule and extended, wherein each adapter of the pair of adapters comprises a UMI, a first or second target specific sequence, and one or more deoxyuridines. 
     
     
         17 . The method of  claim 14 , wherein an adapter has been ligated to the target DNA molecule to generate a ligation product, wherein the adapter comprises one or more deoxyuridines and a unique molecular index (UMI). 
     
     
         18 . The method of  claim 17 , wherein the target DNA molecule has been end-repaired. 
     
     
         19 . The method of  claim 17 or 18 , wherein the target DNA molecule has been adenylated. 
     
     
         20 . The method of any one of  claims 17-19 , wherein the ligating is performed at a pH of 8-9. 
     
     
         21 . The method of any one of  claims 17-20 , wherein the ligating is performed at a PEG concentration of 4% PEG to less than 10% PEG. 
     
     
         22 . The method of any one of  claims 16-21 , wherein the target specific primer comprises a tag sequence comprising at least 2 deoxyuridines and a target specific sequence comprising 0-4 deoxyuridines in a 5′ to 3′ direction. 
     
     
         23 . The method of any one of  claims 14-22 , further comprising treating the target amplification product with a UDG and then amplifying the target amplification product with a universal primer to generate a second target amplification product, wherein the universal primer does not comprise a deoxyuridine. 
     
     
         24 . The method of  claim 23 , wherein the universal primer comprises a sample index. 
     
     
         25 . The method of any one of  claims 14-24 , wherein the amplifying is performed with an antibody based thermostable polymerase. 
     
     
         26 . The method of any one of  claims 21-25 , further comprising purifying the second target amplification product with beads. 
     
     
         27 . The method of  claim 26 , wherein the beads are solid phase reversible immobilization (SPRI) beads. 
     
     
         28 . The method of any one of  claims 14-27 , wherein the DNA library can be used for next generation sequencing, profiling of DNA variants, human identity or paternity testing, pain or ADME pharmacogenomics, or detection of a genetic disease. 
     
     
         29 . A DNA library made by the method of any one of  claims 1-28 . 
     
     
         30 . A kit comprising a uracil-DNA glycosylase (UDG), an adapter comprising one or more deoxyuridines and a unique molecular index (UMI), a universal primer, and an antibody based thermostable polymerase. 
     
     
         31 . The kit of  claim 30 , further comprising a ligase and a ligation buffer capable of providing a pH of 8 to 9 to a ligation reaction. 
     
     
         32 . The kit of  claim 31 , wherein the ligation buffer is capable of providing a PEG concentration of 4% to less than 10% to the ligation reaction. 
     
     
         33 . The kit of any one of  claims 30-32 , further comprising solid phase reversible immobilization (SPRI) beads. 
     
     
         34 . The kit of any one of  claims 30-33 , further comprising a target specific primer comprising one or more deoxyuridines and a target specific sequence,